Soil bacteria of the genus Rhizobium, a member of the family Rhizobiaceae, are capable of infecting plants and inducing a highly differentiated structure known as the root nodule, within which atmospheric dinitrogen is reduced to ammonia by the bacteria. The ammonia is a form of nitrogen readily assimilated by plant tissue. The plant host is most often of the family Leguminosa. In the past Rhizobium species were informally classified in two groups, "slow-growing" or "fast-growing," to reflect growth rates in laboratory culture. The group of "slow-growing" rhizobia has been recently reclassified as a new genus, Bradyrhizobium (Jordan (1982) Internat. J. System. Bacteriol. 32:136; Bergey's Manual of Determinative Bacteriology Vol. 1, 1984, Holt et al., eds). The "fast-growing" members of the genus Rhizobium include the species trifolii, meliloti, leguminosarum, and phaseolus. The Rhizobium species generally display a narrow host range. Fast-growing R. japonicum have also been described which nodulate wild soybean and Glycine max cv. Peking but form ineffective nodules on commercial soybean cultivars. These R. japonicum strains classified as R. fredii in some references, as well as the fast-growing members of the cowpea Rhizobium (now R. loti), display broader host range. The slow-growing rhizobia, now considered a distinct genus called Bradyrhizobium, include the commercially important soybean-nodulating strains B. japonicum (i.e. USDA 110 and USDA 123), the symbiotically promiscuous slow-growing rhizobia of the cowpea group and Bradyrhizobium parasponia, formerly Parasponia Rhizobium. Bradyrhizobium parasponia nodulates a number of tropical legumes including cowpea and siratro, and is distinguished by its ability to nodulate the nonlegume Parasponia.
The genetics of nitrogen fixation have been most extensively studied in the free-living bacterium Klebsiella pneumoniae. A cluster of 17 genes has been identified (Dixon, 1984). Genes with homology to the Klebsiella nif genes (for nitrogen fixation) have been found and characterized in other nitrogen-fixing bacteria including B. japonicum. Other rhizobial genes involved in nitrogen fixation which do not share homology with Klebsiella genes have also been studied, some of which have unassigned function; these are called fix genes. In the rhizobia there are additional genes whose function is required for nodulation; these have been designated nod genes. Information about these genes and their functions has been garnered using modern genetic and molecular biological methods including DNA-DNA hybridization, cloning, DNA sequencing, transposon and site-directed mutagenesis, and DNA and protein mapping.
Nodulation and the development of effective symbiosis is a complex process requiring the participation of both plant and bacterial genes. Several recent reviews of the genetics of the Rhizobium-plant interaction are found in Broughton, ed., (1982) Nitrogen Fixation Volumes 2 and 3 (Clarendon Press, Oxford; Puhler, ed. (1983) Molecular Genetic s of the Bacteria-Plant Interaction (Springer-Verlag, Berlin); Szalay and Legocki, eds. (1985) Advances in Molecular Genetics of the Bacteria-Plant Interaction (Cornell University Publishers, Ithaca, New York); Long (1984) in Plant-Microbe Interactions Volume 1, Kosuge and Nester, eds. (McMillan, New York) pp. 265-306; and Verma and Long, (1983) Internat. Rev. Cytol. (Suppl. 14), Jeon, ed. Academic Press, pp. 211-2445.
The genes required for nodulation and nitrogen fixation in the fast-growing rhizobia have been well-studied. These genes are located on large plasmids called Sym (symbiosis) plasmids in R. meliloti (Kondorosi et al. (1984) Mol. Gen. Genet. 193:445-452), R. trifolii (Schofield (1984) Plant Mol. Biol. 3:3-11), and R. leguminosarum (Downie et al. (1983) Mol. Gen. Genet.190:359-365).
In contrast to the fast-growing rhizobia, no Sym plasmids have been associated with nodulation and nitrogen fixation in the slow-growing rhizobia, B. japonicum or Bradyrhizobium parasponia. The nitrogenase and nodulation genes of these organisms are believed to be encoded on the chromosome. Marvel et al. (1984) in Advances in Nitrogen Fixation Research, Veeger and Newton (eds.) Nijhoff/Junk, The Hague, Netherlands; and Marvel et al. (1985) Proc. Nat. Acad. Sci. USA 82:5841-5845, have shown that a strain of B. parasponia contains genes associated with early nodulation which can functionally complement mutations in R. meliloti nod genes and which can hybridize to the nodABC genes of R. meliloti. The presence of a nodD gene homologue in B. parasponia has been reported. Russell et al. (1985) J. Bacteriol 164:1301-1308 report the isolation of DNA regions encoding nodulation functions in strains of B. japonicum. The isolated DNA region was reported to show strong sequence homology to nod regions of R. meliloti and R. leguminosarum, and to functionally complement a Rod mutant of R. fredii. No sequence data or transcript mapping for the cloned DNA was presented.
In B. japonicum there are two known clusters of genes involved in nodulation and nitrogen fixation. The three structural genes encoding nitrogenase are organized into the nifDK and the nifH operons, which are separated by 17 kbp. A nifE-like gene downstream of nifDK has been found, with interspecific hybridization to nifE of Klebsiella, Sebania Rhizobium, and the R. meliloti fixE gene. A nifB-like gene has been located 11 kbp 3' to nifDK; this gene shows some sequence homology to the corresponding genes of Klebsiella and R. leguminosarum. In contrast to R. meliloti where the three fix genes are in one operon, in B. japonicum they are separated into two operons. fixB and fixC genes are located in the first cluster of genes, and fixA is located in the second cluster near nifA. A nifA-like homologue has been detected in the second cluster of nitrogen fixation-related genes. The postulated function of the gene product of nifA is that of a transcription activator. Nodulation genes have been studied using cloned R. meliloti and R. leguminisarum genes as probes. The nodABC genes are grouped together in B. japonicum as in the fast-growing rhizobia (reviewed in Hennecke et al. (1985) Advances in Nitrogen Fixation Research; Evans et al. (eds.) Martinus Ninjhoff Publishers, Dodrecht, Netherlands, pp. 157-163). A nodD homologue has been found which is unlinked to either gene cluster.
For studies of the genetic organization of the B. japonicum nodulation and nitrogen fixation genes that require recombinant DNA approaches, it has been most efficient that cloning procedures utilize the well-characterized Escherichia coli host system. Transformation of B. japonicum with plasmid DNA has been reported, but it is an inefficient process (Doctor and Modi (1975) in Putnam, ed., Symbiotic Nitrogen Fixation in Plants, International Biological Press, London, pp. 66-76). The more practical way to transfer DNA from E. coli to B. japonicum is by conjugation (Kuykendall (1979) Appl. Environ. Microbiol. 37:862-866; Pilacinski and Schmidt (1981) J. Bacteriol. 145:1025-1030). One can use a self-transmissible plasmid or one can utilize a plasmid system wherein the cloning vector can be mobilized by functions supplied in trans by a second plasmid. The disadvantage of using a self-transmissible plasmid for recombinant DNA work is the large size of most such plasmids.
One example of a broad host range cloning system is the binary vector system of Ditta et al. (1980) Proc. Nat. Acad. Sci.USA 77:7347-7351. The 20 kbp cloning vector pRK290 comprises the broad host range replicon of RK2, a selectable tetracycline resistance gene, EcoRI and BglII cloning sites, and sequences which allow it to be mobilized in trans by functions supplied by a second plasmid. pRK2013 contains the RK2 transfer genes, a kanamycin resistance marker, and the replicon of ColE1, which prevents the maintenance of this plasmid in nonenteric bacteria. pRK290 can be mobilized into a wide range of Gram-negative bacteria with high frequencies. A second example of a cloning system successfully used in the genetic analysis of Rhizobium is with mobilizable vectors developed by Puhler and his co-workers (U.S. Pat. Nos. 4,626,504 and 4,680,264, and U.S. Pat. No. No. 4,686,184.
The disadvantage of introducing cloned DNA into B. japonicum on pRK290 vectors is that although the plasmid replicon is functional, the plasmid is not stably maintained in the absence of selection (Alvarez-Morales et al. (1986) Nucleic Acids Res. 14:4207-4227). Thus experiments with B. japonicum carrying pRK290 derivatives, especially in symbiosis testing, are complicated by plasmid, and therefore cloned gene, loss. Selection for the plasmid also complicates results because tetracycline interferes with the derepression of the nif genes.
Alternative cloning vectors include those derived from other broad host range plasmids of plasmid incompatibility group Q, for example, replicons derived from RSF1010. When the appropriate mobilization sequences are present, those plasmids can also be mobilized in trans by plasmids such as RK2 or pRK2013. Performing much of the work in E. coli has the advantages of a well-studied system, faster growth rate, and a large number of genetic tools available for manipulation of the cloned genes. In many cases, such as for the analysis of mutated DNA for altered nif, nod, or fix gene functions, that cloned DNA must be transferred from E. coli to B. japonicum and integrated into the genome to replace the wild-type copies of those genes.
Integration of cloned DNA carrying a mutation into Rhizobium or Bradyrhizobium has typically been effected by the process of marker exchange, wherein homology between the incoming DNA and the resident chromosomal or plasmid DNA permits recombination so that the mutant copy replaces the genomic copy. pBR325 replicons carrying cloned R. meliloti DNA with Tn5 insertion mutations have been used as vectors mobilized in trans by pRK2013 (Jacobs et al. (1985) J. Bacteriol. 162:469-476). Marker exchange has been a useful tool in the identification and analysis of nodulation and nitrogen fixation genes in the rhizobia, but it is limited in the sense that the cloned gene to be incorporated must be homologous to a region in the genome, and an associated function must be selectable. Alternatively, the technique of marker rescue can be used to select for the replacement of a mutant resident gene with a functional copy of that gene cloned on an incoming plasmid which cannot replicate in that host.
Legocki et al. (1984) Proc. Nat. Acad. Sci. USA 81:5806-5810, have employed a method of random integration for their study of symbiotically regulated promoters. The cloning vector was a suicide vector, capable of being transformed from E. coli to Rhizobium, but incapable of replicating in the nonenteric host. A nif promoter was fused to a .beta.-galactosidase reporter gene, and that recombinant complex was spliced into a random promoterless chromosomal DNA fragment of Rhizobium BTAi1 such that the engineered segment to be incorporated into the genome was flanked by genome-homologous segments of DNA from an unknown, uncharacterized region of the genome. Adjacent to the promoter-reporter gene fusion was a kanamycin resistance gene for selecting a recombinant in which the complex had recombined into the genome. It was possible to obtain recombinants wherein the desired kanamycin resistance gene and the promoter-reporter gene fusion had recombined into the genome with no detectable adverse effects.
Analysis of the B. japonicum genome has revealed the existence of at least five different types of repeated sequences, most of which are located near clusters of genes for nodulation and symbiotic nitrogen fixation (see Hennecke, H. et al. (1987) in Verma, D.P.S. and Brisson, N. (Eds.) Molecular Genetics of Plant-Microbe Interactions; Martinus, Nijhoff Publishers, Dordrecht; pp. 191-196; and Kaluza, et al. (1985) J. Bact. 162:535). The sequences are named RS.alpha., -.beta., -.gamma., .delta., and -.epsilon.. In USDA110, there are 12 copies of RS.alpha., 6 of .beta., and 12, 10 and 4 of .gamma., .delta. and .epsilon., respectively. RS.alpha. and RS.beta. have been characterized by mapping, and restriction analysis. The nucleotide sequence of RS.alpha.9 has been published (Kaluza, et al. 1985). The term, RS elements, is used herein throughout to denote repeated sequences having the following characteristics: they are clustered around the nif region but are not involved in nif-or nod-related functions; they do not contain nif promoter homology; they possess structural characteristics similar to IS elements (potential inverted repeats at their ends, potential target site duplication and containing large open reading frames); and they do not cause significant genome instability. The RS elements are around 1000 bp in length. RS.alpha. has 1126 bp and RS.beta. is 950 bp. The size of RS elements distinguishes them from repeated sequences found in R. trifolii and R. meliloti, which are about 300 bp length (Better, M. et al. (1983), Cell 35:479; Scott, D.B. (1984), Arch. Microbiol. 139:151).
There are two groups of repeated DNA sequence elements of particular value for the present invention in the B. japonicum genome: RS.alpha. and RS.beta.. The RS.alpha. sequence is 1126 bp in length and is repeated 12 times; RS.beta. is about 950 bp in length and is reiterated at least 6 times. Despite the insertion element-like properties, the genomic positions of the RS.alpha. and the RS.beta. elements appear to be quite stable. There are no known functions associated with the RS.alpha. and RS.beta. sequences of B. japonicum. Several copies of both RS.alpha. and RS.beta. are clustered in and around the nif, fix and nod genes. The only location where an RS.alpha. and an RS.beta. sequence are in close proximity to each other is about 450 bp upstream of nifDK (Kaluza et al. (1985) J. Bacteriol. 162;535-542).
Work on which the present application was based was published in an article co-authored by the inventors hereof: G. Acuna, et al. (1987), "A vector for the site-directed, genomic integration of foreign DNA into soybean root nodule bacteria," Plant Molecular Biology 9:41-50, incorporated herein by reference; and in a thesis by G. Acuna for the Microbiology Institute, Swiss Federal Institute of Technology, Zurich, entitled "Construction of a Suitable Vector for the Site-Directed Integration of Foreign DNA Into the Genome of Bradyrhizobium japonicum," also incorporated herein by reference.